| 1 |
GSM31947 |
self-self homotypical DNA CL Brener |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
genomic |
Trypanosoma cruzi
 |
unclassified |
source_name:DNA CL Brener title:self-self homotypical DNA CL Brener description:Self-self (homotypical) control experiment. DNA targets - Whole genomic DNA of CL Brener and Silvio strains was purified. Targets for hybridisation were generated from DNA templates by incorporation of fluorophor-labelled dCTP in a random primer polymerisation reaction. In brief, 50 ml labelling reaction contained 4 mg DNA, 60 mM of either Cy3- or Cy5-dCTP (Amersham Biosciences), 50 mM Tris-HCl pH 6.8, 5 mM MgCl2, 10 mM 2-mercaptoethanol, 0.12 mM each dATP, dGTP, dTTP and 0.06 mM dCTP, 1 ml of random nonamers (Amersham) and 80 U of Klenow DNA polymerase (GIBCO-BRL). Incubation was at 37oC for 2 h. The reaction was stopped by addition of 5 ml 0.5 M EDTA and the products were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting Keywords = homotypical Keywords = LOWESS Keywords = self-self |
| 2 |
GSM31948 |
self-self homotypical DNA CL Brener (replicate) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
genomic |
Trypanosoma cruzi
|
unclassified |
source_name:DNA CL Brener title:self-self homotypical DNA CL Brener (replicate) description:Self-self (homotypical) control experiment. DNA targets - Whole genomic DNA of CL Brener and Silvio strains was purified. Targets for hybridisation were generated from DNA templates by incorporation of fluorophor-labelled dCTP in a random primer polymerisation reaction. In brief, 50 ml labelling reaction contained 4 mg DNA, 60 mM of either Cy3- or Cy5-dCTP (Amersham Biosciences), 50 mM Tris-HCl pH 6.8, 5 mM MgCl2, 10 mM 2-mercaptoethanol, 0.12 mM each dATP, dGTP, dTTP and 0.06 mM dCTP, 1 ml of random nonamers (Amersham) and 80 U of Klenow DNA polymerase (GIBCO-BRL). Incubation was at 37oC for 2 h. The reaction was stopped by addition of 5 ml 0.5 M EDTA and the products were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting Keywords = homotypical Keywords = LOWESS Keywords = self-self |
| 3 |
GSM31949 |
FAMEMA vs. VL10 (bio. repl. 1, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. FAMEMA (isolated from assymptomatic patient) RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 4 |
GSM31950 |
FAMEMA vs. VL10 (bio. repl. 1, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. FAMEMA (isolated from assymptomatic patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 5 |
GSM31951 |
FAMEMA vs. VL10 (bio. repl. 2, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. FAMEMA (isolated from assymptomatic patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 6 |
GSM31952 |
FAMEMA vs. VL10 (bio. repl. 2, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. FAMEMA (isolated from assymptomatic patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 7 |
GSM31953 |
B115 vs. VL10 (bio. repl. 1, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B115 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 8 |
GSM31954 |
B115 vs. VL10 (bio. repl. 1, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B115 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 9 |
GSM31955 |
B115 vs. VL10 (bio. repl. 2, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B115 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 10 |
GSM31956 |
B115 vs. VL10 (bio. repl. 2, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B115 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 11 |
GSM31957 |
B147 vs. VL10 (bio. repl. 1, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B147 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 12 |
GSM31958 |
B147 vs. VL10 (bio. repl. 1, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B147 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 13 |
GSM31959 |
B147 vs. VL10 (bio. repl. 2, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B147 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 14 |
GSM31960 |
B147 vs. VL10 (bio. repl. 2, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B147 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 15 |
GSM31961 |
B13 vs. VL10 (bio. repl. 1, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B13 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 16 |
GSM31962 |
B13 vs. VL10 (bio. repl. 1, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B13 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 17 |
GSM31963 |
B13 vs. VL10 (bio. repl. 2, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B13 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 18 |
GSM31964 |
B13 vs. VL10 (bio. repl. 2, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-07 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. B13 (isolated from cardiac patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 19 |
GSM31965 |
Berenice vs. VL10 (bio. repl. 1, tech repl. 1) |
3,840 |
Universidade de São Paulo |
2004-10-08 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. Berenice (isolated from assymptomatic patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
| 20 |
GSM31966 |
Berenice vs. VL10 (bio. repl. 1, tech repl. 2) |
3,840 |
Universidade de São Paulo |
2004-10-08 |
[cDNA Array] cDNA microarray Trymanosoma cruzi - v.2 (GPL1484) |
RNA |
Trypanosoma cruzi
|
bone marrow |
VL10 (isolated from assymptomatic patient) vs. Berenice (isolated from assymptomatic patient). RNA targets - 5 x 108 parasite cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to the manufacturer's protocol. RNA was DNase-treated and approximately 20 mg was labelled with fluorophor-labelled dCTP in the first-strand cDNA synthesis. The reaction mix contained 0.05 mM of either Cy3- or Cy5-dCTP, 1 ml oligo-dT(15) primer (Amersham Biosciences), 4 ml of random nonamers (Amersham), 0.1 mM each of dATP, dCTP, dTTP and 0.05 mM dCTP, 10 mM dithiothreitol and 400 units of Superscript II Reverse Trascriptase (Invitrogen) in the buffer provided by the manufacturer. Incubation was performed at 42oC for 2.5 h. Subsequently, RNA was hydrolysed by addition of 2 ml 2.5 M NaOH and an incubation at 37ºC for 15 min. The solution was neutralised by addition of 10 ml 2 M Hepes. The samples were purified with Multiscreen plates (Millipore). The incorporation was determined by photometric measurement (550 nm for Cy3 and 650 nm for Cy5). The samples were dried in vacuum and dissolved in H2O to reach 25% of the solution plus 25% hybridisation buffer (Amersham Pharmacia) and 50% formamide. DNA was denatured at 98oC for 2 min. Hybridisation to the array was performed under a glass cover-slide (24 x 60 mm). The slides were kept in a waterproof hybridisation chamber in a 42oC water bath for at least 16h. After hybridisation, slides were washed with 1 x SSC, 0.2% SDS, followed by two washes in 0.1 x SSC, 0.2% SDS. All washings were performed at 55oC for 10 min. An additional wash was performed at RT in 0.1 x SSC for one min. Slides were dried and subjected to fluorescence detection. Image analysis - The slides were scanned with a laser scanner (Molecular Dynamics) resulting in 16-bit images. Intensities were extracted using ArrayVision 6.0 (Image Research) software. Normalisation of the hybridisation data was achieved by LOWESS fitting. Keywords = strains comparison |
|